Dihydroethidium (DHE) is a fluorescent probe used to detect the generation of superoxide anions (O₂•⁻) within cells; it is able to penetrate the cell membrane and enter the cell. Upon reaction with superoxide, dihydroethidium forms a red fluorescent product (Ex = 500–530 nm / Em = 590–620 nm), which can be used for the detection and imaging of intracellular superoxide.

METHODS: Detection of cellular peroxides by Dihydroethidium:
1. Prepare a 5-10 mM stock solution of Dihydroethidium dye in DMSO, protect from light, and store in portions at -20℃.
Dilute the stock solution with physiological buffers (e.g., PBS, HBSS, HEPES) to obtain a working solution of 5-20 µM Dihydroethidium dye. The optimal working concentration must be determined empirically.
3. Add the Dye Working Solution to the cells at a 1:1 ratio (100 µL of Dye Working Solution to 100 µL of Cell Suspension) and incubate for 5-60 minutes at room temperature or 37℃.
4. Wash with PBS and then perform fluorescence microscopy or flow cytometry. Dihydroethidium itself is blue fluorescent with a maximum excitation wavelength of 370 nm and a maximum emission wavelength of 420 nm, and after dehydrogenation, it binds to RNA or DNA to produce red fluorescence with a maximum excitation wavelength of 300 nm and a maximum emission wavelength of 610 nm, and 518 nm can be used as the excitation wavelength in practice.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

Dihydroethidium detection of cell peroxides
1. Prepare 5-10 mM Dihydroethidium dye stock solution with DMSO, protect from light, and store in aliquots at -20°C.
2. Dilute the stock solution with physiological buffer (such as PBS, HBSS, HEPES) to obtain 5-20 µM Dihydroethidium dye working solution. The optimal working concentration must be determined empirically.
3. Add the dye working solution to the cells at a ratio of 1:1 (100 µL cell suspension to 100 µL dye working solution) and incubate at room temperature or 37°C for 5-60 minutes.
4. After washing with PBS, perform fluorescence microscopy or flow cytometry detection. Dihydroethidium itself is a blue fluorescent substance with a maximum excitation wavelength of 370 nm and a maximum emission wavelength of 420 nm. After dehydrogenation, it combines with RNA or DNA to produce red fluorescence with a maximum excitation wavelength of 300 nm and a maximum emission wavelength of 610 nm. In actual observation, 518 nm can also be used as the excitation wavelength.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

DMSO: 125 mg/mL (396.31 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 0.5 mg/mL (1.59 mM), Solution.